Neutrophils (PMNs) are phagocytic cells that produce superoxide (O2-) needed for host defense, but under specific circumstances O2- production can also result in tissue damage such as found in Acute Lung Injury. Our current understanding of the PMN nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is based primarily on studies using phorbol esters. Chemoattractants such as N-formyl-Met-Leu-Phe (fMLF) induce O2- generation in PMNs, yet the underlying mechanisms remain poorly understood. A novel, whole cell-based reconstitution system, using COS-phox cells, has been developed to address, in a systematic manner, the signaling mechanisms mediating NADPH oxidase activation. These studies will be complemented with experiments using wild-type PMNs and PMNs from mice with gene deletions, as well as experiments addressing how the activation of specific signaling pathways induces lung vascular injury, or may be protective in other instances. Aim 1 is based on our preliminary findings that PKCzeta is sufficient for reconstituting the fMLF-induced O2- production in COS-phox cells. Thus, we will test the hypothesis that PKCzeta plays a critical role in fMLF activation of NADPH oxidase and identify the phosphorylation steps involved. In Aim 2, we will extend our finding that p40phox selectively enhances fMLF- but not PMA-induced O2- generation, and will test the hypothesis that p40phox plays an important role in regulating the signaling pathways mediating NADPH oxidase activation, in addition to its role in NADPH oxidase complex formation. In Aim 3, we will test the hypothesis that G-alpha-q-mediated PLCzeta activation (as induced by the PMN priming action of platelet-activating factor) sensitizes the enzyme for subsequent activation by heterotrimeric G protein beta-gamma subunits, which are released upon fMLF-stimulation of its G protein coupled receptor (GPCR), FPR. In Aim 4, we will determine the function of the novel lipid mediator lysophosphatidylcholine (LPC) in down-regulating NADPH oxidase and address the role of LPC in preventing PMN-mediated lung vascular injury. We will test the hypothesis that LPC binding to its G-alpha-s-coupled receptor G2A leads to a rise in intracellular cAMP that thereby inhibits PMN O2- production. By incorporating cellular and molecular studies into the study of microvascular permeability in mouse lung models, we will gain a better understanding of the mechanisms of NADPH oxidase activation by GPCRs and how NADPH oxidase activation can be modulated. With the insights gained, we will be in a position to develop novel therapeutic strategies targeting inappropriate PMN activation and PMN-mediated lung vascular injury and edema.